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Abstract

The targeted inactivation of a single oncogene can induce dramatic tumor regression,
suggesting that cancers are “oncogene addicted.” Tumor regression following oncogene
inactivation has been thought to be a consequence of restoration of normal physiological
programs that induce proliferative arrest, apoptosis, differentiation, and cellular
senescence. However, recent observations illustrate that oncogene addiction is highly
dependent upon the host immune cells. In particular, CD4+ helper T cells were shown to be essential to the mechanism by which MYC or BCR-ABL
inactivation elicits “oncogene withdrawal.” Hence, immune mediators contribute in
multiple ways to the pathogenesis, prevention, and treatment of cancer, including
mechanisms of tumor initiation, progression, and surveillance, but also oncogene inactivation-mediated
tumor regression. Data from both the bench and the bedside illustrates that the inactivation
of a driver oncogene can induce activation of the immune system that appears to be
essential for sustained tumor regression.

Keywords:

Oncogene addiction; MYC; Tumor microenvironment; Tumor immunology

Introduction

Capitalizing on oncogene addiction: a therapeutic objective

The inactivation of a single oncogene can result in the dramatic and sustained regression
of some cancers [1-4]. Targeted inactivation of an oncogene can be associated with proliferative arrest,
apoptosis and/or senescence, and differentiation [3]. Oncogene addiction appears to be a consequence of the restoration of physiological
programs [2,5], but also has been described as a consequence of synthetic lethality [6] and the differential decay of survival and apoptosis programs [7]. “Oncogene withdrawal” occurs upon suppression of initiating genetic events in tumors
[8,9]. It is not known when a cancer will be addicted to a particular oncogene [4]. Oncogene addiction has been thought to occur through host cell autonomous, tumor
intrinsic mechanisms. Yet, recent observations illustrate that oncogene addiction
has both cell autonomous as well as immune-mediated mechanisms [10-14] (Figure 1).

Figure 1.The host immune system is required for sustained tumor regression following oncogene
withdrawal. Following oncogene inactivation in a mouse model by transgenic methods or in patients
by oncogene-targeted therapy, there are tumor cell-intrinsic consequences, immunological
consequences, and host microenvironmental consequences. Tumor cell-intrinsic consequences
include proliferative arrest and the induction of apoptosis. Dying tumor cells and
antigen debris may stimulate an immune response, which may in turn feed back in to
the tumor cell-intrinsic consequences. The immune response, particularly helper T
cells, can influence environmental consequences, including the induction of senescence
and the collapse of angiogenesis. Lastly, senescing tumor cells may have a secretory
phenotype, which in turn may influence the immune system. Taken together, these three
components lead to a remodeling of the entire tumor (both in the cancer cells and
in the environment) and contribute to lasting tumor regression and protection from
relapse.

Oncogene addiction has been largely studied in mouse models. However, tumor regression
following oncogene inactivation has been observed in response to targeted therapeutics
in humans including molecules that target BCR-ABL or c-Kit, EGFR, ALK, BRAF V600E,
PML-RARα, and HER2/neu for the treatment of leukemia, lung adenocarcinoma, non-small
cell lung cancer, melanoma, and breast cancer [15-23]. Other drugs are in clinical investigation, including those that target JAK2, MDM2,
and PI3 Kinase [24-31]; drugs that target RAS [32] and MYC [33,34] are in early development. It remains to be seen if these agents specifically take
advantage of oncogene addiction. In general, little has been done to study the mechanism
of action of these therapeutic agents in human patients.

Experimental mouse models have been a particularly tractable approach to interrogating
the mechanism of oncogene addiction. Transgenic mouse models employing strategies
that enable the conditional expression of oncogenes have been used to illustrate that
cancers initiated by an oncogene, such as MYC, RAS, BCR-ABL, MET, and BRAF, are reversible
upon suppression of the oncogene [1,35-40].

Review

Cancer and the immune system: a complex relationship

The mechanisms by which targeted therapies engage oncogene addiction have been presumed
to be cell autonomous. However, oncogene inactivation causes dramatic changes in the
microenvironment including the shut down of angiogenesis [41-44] and the recruitment of host effector cells, including innate and adaptive immune
cells [4,45-48]. Thus it appeared likely that the immune system is playing an active role in the
mechanism of tumor regression following oncogene inactivation.

Furthermore, it is well known that the immune system is a barrier to tumorigenesis
[49]. Hosts with absent or suppressed immune systems have a greatly increased incidence
of many different types of cancer [50,51]. Patients who are transplant recipients and take drugs that suppress their adaptive
immunity demonstrate dramatically increased incidences of lymphoma and squamous cell
carcinoma [52-54]. Moreover, patients who are immunosuppressed also have an impeded response to cancer
therapy with a decreased overall and progression-free survival [55,56]. Thus, immune surveillance mechanisms are critical both to the prevention as well
the efficacy of conventional treatment of these cancers [57-59] and are a critical component to the therapeutic efficacy of agents for cancer [54,60-62].

Correspondingly, the activation of the immune system through specific immune-based
therapies is efficacious for the treatment of some cancers. This includes antibodies
that target cancer cells, such as Rituximab [63] and Trastuzumab [64], as well as antibodies or drugs that modulate immunostimulatory or immunoinhibitory
signals [65-67], such as anti-CTLA-4 [68] and anti-PD-L1 [69]. The combination of conventional chemotherapy with targeted immune therapy has emerged
as an effective approach for the treatment of some cancers.

Oncogene inactivation activates the immune system

Recent studies in experimental mouse models illustrate the mechanisms by which oncogene
“withdrawal” results in immune activation (Figure 1, [24,45]). In a tetracycline-regulated conditional mouse model of MYC-induced T cell Acute
Lymphoblastic Leukemia (T-ALL), the tumor cells undergo proliferative arrest and death
within 2 days of turning off the MYC oncogene via tumor intrinsic, host independent,
immune independent mechanisms. Subsequently, between 2 and 5 days, there is a recruitment
of immune effector cells that are required to induce cellular senescence of tumor
cells and the shut down of angiogenesis in the tumor microenvironment [70]. The kinetics of tumor regression, the extent of tumor regression, and the ability
to maintain sustained tumor regression are all compromised in immunodeficient hosts.

Provocatively, CD4+ helper T cells were found to be the key immune effector required for oncogene inactivation-induced
tumor regression in the conditional MYC-driven T-ALL mouse model. The CD4+ T cells are likely to contribute to tumor regression through many mechanisms. Of
note, CD4+ T cells can express a variety of cytokines that have been implicated in the regulation
of cellular senescence and/or angiogenesis [71-74]. CD4+ T cells may also be working via direct cellular interactions with the tumor cells
or host stromal cells in the tumor microenvironment. Finally, CD4+ T cells appear to recruit other immune and host cells.

The CD4+ helper T cells must express thrombospondins in order to contribute to tumor regression
following oncogene inactivation [45]. TSP-1 has been suggested to be a key regulator of both angiogenesis and senescence
[75]. Moreover, CD47, the receptor of TSP-1, is a key regulator of the immune response
[76]. TSP-1 and CD47 have been suggested to regulate cellular senescence [75,77,78]. However, there is also a general induction of anti-tumor and a suppression of pro-tumor
cytokines after oncogene inactivation that occurs only in immunocompetent hosts [45]. Hence, specific secreted factors are likely to contribute to the mechanism of oncogene
addiction and withdrawal.

How oncogene inactivation recruits a response of CD4+ T cells is not known. There are several possibilities. First, oncogenes such as MYC
have been suggested to regulate the expression of molecules that may be immunosuppressive
and/or regulate angiogenesis. Hence, MYC inactivation could lead to the direct change
in expression of cytokines by tumor cells, thereby recruiting immune cells [79]. Second, oncogene inactivation could activate an immune response through immunogenic
cell death that in turns activates the immune response [80]. Identifying the specific mechanism of the immune activation and response could suggest
important strategies for monitoring and implementing a therapeutic response [10].

Importantly, many other immune effectors are likely to contribute to the response
of targeted therapies. This is potentially governed by the unique genetic and cellular
context of each tumor [81,82]. In other mouse models, investigators have noted that innate immune cells such as
mast cells [83], macrophages [84], and other antigen-presenting cells (APCs) may function as barriers to tumor growth
and facilitators of tumor regression. Thus, it is likely that these other innate and
adaptive immune cells contribute to the mechanism of oncogene addiction and tumor
regression following oncogene inactivation.

In the clinic: targeted oncogene inactivation and immune response

Oncogene addiction has been studied in a more limited manner in human patients. Some
studies indicate that the host immune response is essential for the optimal response
to conventional chemotherapy and radiotherapy [85-87]. A major potential limitation of conventional therapeutics is that they often suppress
the immune response [88].

Other correlative studies suggest that an immune response may contribute to the mechanism
of targeted oncogene inactivation. In human patients with BCR-ABL+ gastrointestinal stromal tumors (GIST) treated with Imatinib, IFN-γ secretion by
NK cells in the peripheral blood is associated with a better clinical response [89]. Similarly, the inhibition of BRAF both directly inhibits tumor growth but also appears
to activate the immune system [90]. The combination of a BRAF inhibitor, Vemurafenib, with immune therapy may be more
effective in the treatment of tumors [91]. Moreover, Vemurafenib was associated with intratumoral accumulation of adoptively
transferred T cells [92] as well as increased intratumoral numbers of CD4+ or CD8+ T cells [90] and this was associated with a better prognosis [90]. Other studies have shown that BRAF inhibition is associated with the reduction of
immunosuppressive cytokines and chemokines [93]. Ongoing clinical studies are examining if Vemurafenib in combination with immunotherapy
is more clinically effective [94,95].

Other targeted therapies may induce an immune response in addition to their tumor-specific
effects. Sunitinib, which targets PDGFR, RET, and KIT, recruits an immune response
that may contribute to its mechanism [96] through the induction of IFN-γ-producing T cells [97] and decreased regulatory T cells [97,98]. Arsenic and all-trans-retinoic-acid (ATRA), used for the treatment of PML-RARα acute
promylecytic leukemia, is associated with altered antigen presentation [99]. Bortezomib is a proteasome inhibitor used in the treatment of hematopoietic tumors
and is associated with the recruitment to tumor sites of CD8+ T cells and dendritic cells [100]. The EGFR inhibitor, Erlotinib, is effective in the treatment of non-small cell lung
cancer and is associated with increased intratumoral numbers of dendritic cells [101]. Trastuzumab targets HER2/neu for the treatment of breast and ovarian cancer and
may require an NK cell response [102,103]. Thus, targeted inhibition of oncogenes may be efficacious in part through the activation
of an immune response.

In some cases, targeted inactivation of oncogenes could inhibit an immune response
and impede the efficacy of an anti-tumor therapeutic. For example, inhibition of MAPK/extracellular
signal-regulated kinase kinase (MEK) results in T cell inhibition [104]. Imatinib can affect the immune response in a multitude of ways [24,45,105-109]. Thus, it will be pivotal to consider how targeted oncogene inactivation can induce
or suppress an immune response and how this may contribute to the mechanism of action
of anti-neoplastic agents.

Therapeutic implications for oncogene-targeted therapies

Experimental evidence and clinical observations suggest that targeted oncogene inactivation
generates an anti-tumor immune response. More generally this suggests that targeted
oncogene inactivation can be exploited as an immune therapy. Unlike conventional chemotherapy
or radiotherapy, the judicious choice of agents that target specific oncogenes may
lead to tumor regression both by directly targeting tumor cells and indirectly by
inducing a robust immune response. If this were the case, it would have several practical
implications for the development and application of therapeutics.

First, the combination of oncogene-targeted therapy with specific immunomodulatory
therapy may further increase the clinical response and long-term survival of patients
[94,110,111]. Pointedly, immune activation may be essential to prevent the emergence of therapy-resistant
tumor cells, which can lead to tumor recurrence [112,113]. Hence, the identification of the best agents to prompt oncogene withdrawal will
require examination of the efficacy of these therapies with consideration of their
ability to induce both cell autonomous and host-dependent mechanisms of tumor regression.

Several targeted therapies are currently approved or under investigation in combination
with immunomodulatory therapies (Table 1). For the treatment of melanoma, MEK and VEGF inhibitors are being administered with
Ipilimumab [114,115] and IL-2 [116], respectively. BRAF inhibitors are being examined together with Ipilimumab [117]. Ipilimumab is also being interrogated in combination with Brentuximab for the treatment
of Hodgkin’s Lymphoma [118] and with Crizotinib for non-small cell lung cancer [119]. Ipilimumab and anti-PD-L1 inhibitors are being analyzed in combination with Erlotinib
in non-small cell lung cancer [119,120].

Targeted therapies together with immune-based therapies are also being examined for
the treatment of other types of cancer. Lenalidomide and Bortezomib are being examined
for treatment of multiple myeloma [121], Lenalidomide and Ibrutinib are under investigation for Chronic Lymphocytic Leukemia
[122], and Nivolumab is being administered with Sunitinib for renal cell cancer [123]. Additionally, the mTOR inhibitor Temsirolimus is being studied with Interferon-α
for renal cancer [124]. Imatinib and Rituximab are being investigated in combination with Nivolumab [125] or Pidilizumab [126]. Trastuzumab is under investigation with peptide vaccines and cytokines [127]. These investigations may identify combinations of targeted and immune-based therapies
that are more efficacious for the treatment of cancer. Furthermore, the appreciation
that immune activation may be a critical component to the efficacy of therapeutics
may be important for the measurement and maximization of their clinical efficacy.

Table 1.Targeted therapies studied or under investigation in cooperation with immune therapies

Conclusions

Experimental and clinical observations suggest a model of oncogene addiction and a
role for the immune system (Figure 1). The inactivation of an oncogene in a tumor appears to initiate cancer cell-intrinsic
programs of tumor regression including proliferative arrest, differentiation, and
apoptosis, as well as immune-dependent modulation of the microenvironment that contributes
to cellular senescence and the shut down of angiogenesis. These mechanisms are collectively
required for complete and sustained tumor regression.

Oncogene inactivation in a tumor results in activation of an immune response (Figure 1). The mechanisms by which this occurs are not defined. These mechanisms potentially
involve both direct mechanisms related to the production of immune recruiting cytokines
as well as more indirect mechanisms such as immunogenic cell death. Many cellular
and cytokine effectors are likely to be involved, including CD4+ T cells, CD8+ T cells, B cells, and innate immune cells such as macrophages and NK cells (Figure 1). It is possible that the impairment of specific cellular, humoral, or chemokine
mechanisms would facilitate the re-emergence of tumor cells that are refractory to
targeted therapy.

There are several practical implications of this model. First, successful targeted
therapy against a cancer is likely to require an intact host immune system. Second,
the measurement of the efficacy of a targeted therapy is likely to be most readily
defined through interrogation of immune activation after drug administration. Third,
the early development of therapeutic agents should be performed using model systems
that have an intact host immune system as opposed to in vitro model systems or xenograft
model systems in severely immunocompromised animals.

Our model predicts that the immune system not only directly eliminates tumor cells
but also plays a critical role in modulating the tumor microenvironment. Diagnostic
assays that detect an immune response may predict the therapeutic efficacy of oncogene-targeted
agents. Strategies need to be developed that would enable the measurement of these
effector cells and molecules before and after therapeutic treatment. This could include
in situ measurements in patients using flow cytometry analysis of immune effector
cells, proteomic and genomic analysis, and noninvasive molecular imaging methods.

Finally, the most effective clinical strategy to treat tumors will likely require
a coordination of therapies that target oncogenes in combination with the activation
of specific immune effectors. Conversely, existing conventional chemotherapies that
often impede an immune response may antagonize the efficacy of targeted therapeutics.
Hence, mechanistic insight into how oncogene withdrawal prompts immune activation
may actualize rationale therapeutic strategies.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

SCC and DWF conceived of and wrote the review. YL helped write the review. ACF provided
a clinical perspective on targeted and immunological therapies. All authors read and
approved the final manuscript.

Acknowledgements

The authors acknowledge current and former members of the Felsher laboratory. Within
the Felsher laboratory, research has been funded by the Burroughs Welcome Fund Career
Award, the Damon Runyon Foundation Lilly Clinical Investigator Award, NIH NCI K23
CA140722 to A.C.F., NIH RO1 grant number CA 089305, 105102, 170378 PQ22, U54CA149145,
U54CA143907, National Cancer Institute’s In-vivo Cellular and Molecular Imaging Center
grant number CA 114747, Integrative Cancer Biology Program grant number CA 112973,
NIH/NCI PO1 grant number CA034233, and the Leukemia and Lymphoma Society Translational
Research grant number R6223-07. S.C.C. was previously supported by NIH, 5 T32 AI07290
and is currently supported by an NIH NRSA from the NCI (F32CA177139).

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